Over the past 30 years, extensive experimental evidence has accumulated supporting the implication of oxidative stress in the pathogenesis of aging and cardiovascular diseases (CVD) [1-3]. Admittedly, randomized clinical trials with “natural antioxidants” have been disappointing (HOPE, HPS, GISSI-prevention) and have led some to question the relevance of the oxidative stress hypothesis [4-6]. However, the majority of these studies did not evaluate the impact of the antioxidant intervention on the oxidative stress status, or used the highly criticized thiobarbituric acid reactive substances method (TBARS) [7-9]. This can be explained in part by difficulties encountered in validating methods to assess oxidative stress biomarkers in accessible fluids for human studies, which often require expensive and complex mass spectrometric technologies. Recently, isoprostanes have emerged as relatively good markers of oxidative stress-induced lipoperoxidation in vivo [7,9]. However, the measurement of a single biomarker is unlikely to provide a comprehensive picture of the various oxidative stress related events that may contribute to CVD progression.
One oxidative stress-related molecule that has generated considerable research interest over the past 10 years [10] is 4-hydroxy-2,3-nonenal (HNE). HNE is an aldehyde end-product generated by peroxidation of the most abundant class of n-6 polyinsaturated fatty acids [11]. Similar to free radicals, aldehydes are electrophile that react readily to nucleophilic residues of proteins, nucleic acids and lipids, but their relatively longer half-life make them candidates for the propagation of the damage to neighboring cells. Among the aldehydes, 4-hydroxy-2-alkenals such as HNE are considered the most reactive species because of their α,β-double bond [11].
The interest for HNE stems not only from its potential use as a biomarker of oxidative stress-induced lipid peroxidation (LPO), but also because of accumulating evidence indicating that HNE is able to modulate signaling pathways involved in cell proliferation, apoptosis and inflammation, which are hallmarks of CVD [12, 13]. However, much remains to be learned on the role of HNE as an active biomarker of oxidative stress-related events in CVD. Because of the rapid cellular metabolism of HNE, through either reduction to 1,4-dihydroxynonene (DHN), oxidation to 4-hydroxynonenoic acid, or conjugation with glutathione [14], recent studies have highlighted the potential usefulness of measuring HNE metabolites such as dihydroxynonene mercapturic acid in urine [15] or in plasma [16], rather than free HNE.
HNE-protein adducts have also been identified. Increased levels of these adducts, assessed by immunological and gas chromatography-mass spectrometry (GCMS) methods, were reported under conditions of oxidative stress in myocardial tissues [17-20], in circulating albumin [21] and oxidized lipoproteins [22]. However, the possibility that circulating HNE-protein adducts could reflect enhanced systemic or tissue-specific oxidative stress has not been previously examined, and methods of quantifying these adducts in whole blood, plasma or other blood derivatives samples has not been previously described.
Thus, while oxidative stress has been implicated in numerous degenerative diseases of aging, including cardiovascular diseases, there is still a need to identify biomarkers of oxidative stress-related events, such as the lipid peroxidation product 4-hydroxy-2,3-nonenal (HNE) and protein thioether adducts thereof, in these diseases, and particularly in humans. In view of the above, there is a need in the industry to provide novel methods for detecting and quantifying oxidative stress in a biological sample through the use of suitable biomarkers.